Associations of Testosterone and Related Hormones With All-Cause and Cardiovascular Mortality and Incident Cardiovascular Disease in Men : Individual Participant Data Meta-analyses.

BACKGROUND: Whether circulating sex hormones modulate mortality and cardiovascular disease (CVD) risk in aging men is controversial. PURPOSE: To clarify associations of sex hormones with these outcomes. DATA SOURCES: Systematic literature review to July 2019, with bridge searches to March 2024. STUDY SELECTION: Prospective cohort studies of community-dwelling men with sex steroids measured using mass spectrometry and at least 5 years of follow-up. DATA EXTRACTION: Independent variables were testosterone, sex hormone-binding globulin (SHBG), luteinizing hormone (LH), dihydrotestosterone (DHT), and estradiol concentrations. Primary outcomes were all-cause mortality, CVD death, and incident CVD events. Covariates included age, body mass index, marital status, alcohol consumption, smoking, physical activity, hypertension, diabetes, creatinine concentration, ratio of total to high-density lipoprotein cholesterol, and lipid medication use. DATA SYNTHESIS: Nine studies provided individual participant data (IPD) (255 830 participant-years). Eleven studies provided summary estimates (n = 24 109). Two-stage random-effects IPD meta-analyses found that men with baseline testosterone concentrations below 7.4 nmol/L (<213 ng/dL), LH concentrations above 10 IU/L, or estradiol concentrations below 5.1 pmol/L had higher all-cause mortality, and those with testosterone concentrations below 5.3 nmol/L (<153 ng/dL) had higher CVD mortality risk. Lower SHBG concentration was associated with lower all-cause mortality (median for quintile 1 [Q1] vs. Q5, 20.6 vs. 68.3 nmol/L; adjusted hazard ratio [HR], 0.85 [95% CI, 0.77 to 0.95]) and lower CVD mortality (adjusted HR, 0.81 [CI, 0.65 to 1.00]). Men with lower baseline DHT concentrations had higher risk for all-cause mortality (median for Q1 vs. Q5, 0.69 vs. 2.45 nmol/L; adjusted HR, 1.19 [CI, 1.08 to 1.30]) and CVD mortality (adjusted HR, 1.29 [CI, 1.03 to 1.61]), and risk also increased with DHT concentrations above 2.45 nmol/L. Men with DHT concentrations below 0.59 nmol/L had increased risk for incident CVD events. LIMITATIONS: Observational study design, heterogeneity among studies, and imputation of missing data. CONCLUSION: Men with low testosterone, high LH, or very low estradiol concentrations had increased all-cause mortality. SHBG concentration was positively associated and DHT concentration was nonlinearly associated with all-cause and CVD mortality. PRIMARY FUNDING SOURCE: Medical Research Future Fund, Government of Western Australia, and Lawley Pharmaceuticals. (PROSPERO: CRD42019139668).

Factors Associated With Circulating Sex Hormones in Men : Individual Participant Data Meta-analyses.

BACKGROUND: Various factors modulate circulating testosterone in men, affecting interpretation of testosterone measurements. PURPOSE: To clarify factors associated with variations in sex hormone concentrations. DATA SOURCES: Systematic literature searches (to July 2019). STUDY SELECTION: Prospective cohort studies of community-dwelling men with total testosterone measured using mass spectrometry. DATA EXTRACTION: Individual participant data (IPD) (9 studies; n = 21 074) and aggregate data (2 studies; n = 4075). Sociodemographic, lifestyle, and health factors and concentrations of total testosterone, sex hormone-binding globulin (SHBG), luteinizing hormone (LH), dihydrotestosterone, and estradiol were extracted. DATA SYNTHESIS: Two-stage random-effects IPD meta-analyses found a nonlinear association of testosterone with age, with negligible change among men aged 17 to 70 years (change per SD increase about the midpoint, -0.27 nmol/L [-7.8 ng/dL] [CI, -0.71 to 0.18 nmol/L {-20.5 to 5.2 ng/dL}]) and decreasing testosterone levels with age for men older than 70 years (-1.55 nmol/L [-44.7 ng/dL] [CI, -2.05 to -1.06 nmol/L {-59.1 to -30.6 ng/dL}]). Testosterone was inversely associated with body mass index (BMI) (change per SD increase, -2.42 nmol/L [-69.7 ng/dL] [CI, -2.70 to -2.13 nmol/L {-77.8 to -61.4 ng/dL}]). Testosterone concentrations were lower for men who were married (mean difference, -0.57 nmol/L [-16.4 ng/dL] [CI, -0.89 to -0.26 nmol/L {-25.6 to -7.5 ng/dL}]); undertook at most 75 minutes of vigorous physical activity per week (-0.51 nmol/L [-14.7 ng/dL] [CI, -0.90 to -0.13 nmol/L {-25.9 to -3.7 ng/dL}]); were former smokers (-0.34 nmol/L [-9.8 ng/dL] [CI, -0.55 to -0.12 nmol/L {-15.9 to -3.5 ng/dL}]); or had hypertension (-0.53 nmol/L [-15.3 ng/dL] [CI, -0.82 to -0.24 nmol/L {-23.6 to -6.9 ng/dL}]), cardiovascular disease (-0.35 nmol/L [-10.1 ng/dL] [CI, -0.55 to -0.15 nmol/L {-15.9 to -4.3 ng/dL}]), cancer (-1.39 nmol/L [-40.1 ng/dL] [CI, -1.79 to -0.99 nmol/L {-51.6 to -28.5 ng/dL}]), or diabetes (-1.43 nmol/L [-41.2 ng/dL] [CI, -1.65 to -1.22 nmol/L {-47.6 to -35.2 ng/dL}]). Sex hormone-binding globulin was directly associated with age and inversely associated with BMI. Luteinizing hormone was directly associated with age in men older than 70 years. LIMITATION: Cross-sectional analysis, heterogeneity between studies and in timing of blood sampling, and imputation for missing data. CONCLUSION: Multiple factors are associated with variation in male testosterone, SHBG, and LH concentrations. Reduced testosterone and increased LH concentrations may indicate impaired testicular function after age 70 years. Interpretation of individual testosterone measurements should account particularly for age older than 70 years, obesity, diabetes, and cancer. PRIMARY FUNDING SOURCE: Medical Research Future Fund, Government of Western Australia, and Lawley Pharmaceuticals. (PROSPERO: CRD42019139668).

Serum DHEA and Testosterone Levels Associate Inversely With Coronary Artery Calcification in Elderly Men.

CONTEXT: Epidemiological and preclinical data support cardiovascular, mainly protective, effects of sex steroids in men, but the mechanisms underlying the cardiovascular actions of sex steroids are poorly understood. Vascular calcification parallels the development of atherosclerosis, but is increasingly recognized as a diversified, highly regulated process, which itself may have pathophysiological importance for clinical cardiovascular events. OBJECTIVE: To investigate the association between serum sex steroids and coronary artery calcification (CAC) in elderly men. METHODS: We used gas chromatography tandem mass spectrometry to analyze a comprehensive sex steroid profile, including levels of dehydroepiandrosterone (DHEA), androstenedione, estrone, testosterone, estradiol, and dihydrotestosterone, in men from the population-based AGES-Reykjavik study (n = 1287, mean 76 years). Further, sex hormone-binding globulin (SHBG) was assayed and bioavailable hormone levels calculated. CAC score was determined by computed tomography. The main outcome measures were cross-sectional associations between dehydroepiandrosterone, androstenedione, estrone, testosterone, dihydrotestosterone, and estradiol and quintiles of CAC. RESULTS: Serum levels of DHEA, androstenedione, testosterone, dihydrotestosterone, and bioavailable testosterone showed significant inverse associations with CAC, while estrone, estradiol, bioavailable estradiol, and SHBG did not. DHEA, testosterone, and bioavailable testosterone remained associated with CAC after adjustment for traditional cardiovascular risk factors. In addition, our results support partially independent associations between adrenal-derived DHEA and testes-derived testosterone and CAC. CONCLUSION: Serum levels of DHEA and testosterone are inversely associated with CAC in elderly men, partially independently from each other. These results raise the question whether androgens from both the adrenals and the testes may contribute to male cardiovascular health.

Sex hormone-dependent and -independent regulation of serum BAFF and TNF in cohorts of transgender and cisgender men and women.

Background: The risk of inflammatory diseases is sex-dependent, but it remains unknown whether this is due to the impact of sex hormones or sex chromosomes. Transgender individuals represent a unique cohort for studying the relative influence of endocrine and chromosomal factors. Here we compared serum levels of B-cell activating-factor (BAFF) and tumor necrosis factor (TNF) in transgender men (TM), transgender women (TW), cisgender women (CW) and cisgender men (CM). Methods: BAFF and TNF were measured in the serum of 26 CW, 30 CM, 27 TM and 16 TW individuals. To determine the responsiveness of immune cells, TNF was measured in bacterial lipopolysaccharide (LPS)-treated peripheral leukocytes. Results: BAFF was higher in CF (998 pg/mL) and TW (973 pg/mL) compared to CM (551 pg/mL) (P < 0.0001) and TM (726 pg/mL) (P < 0.0001). No difference in BAFF levels was shown between subjects grouped according to the number of X chromosomes. TNF was higher in CM (174 pg/mL) than TW (2.3 pg/mL) (P = 0.027) and TM (27.4 pg/mL) (P = 0.028). LPS-induced TNF was higher in CM (2524 pg/mL) and TM (2078 pg/mL) than in CW (1332 pg/mL) (both P < 0.0001) and TW (1602 pg/mL) (both P = 0.009). Discussion: Sex hormones and sex chromosomes have different impacts on cytokines involved in the sex-dependent inflammatory response. The concentration of BAFF and LPS-stimulated TNF secretion depended on sex hormone levels, whereas basal TNF was regulated by both sex hormone-dependent and -independent factors.

Castration of Male Mice Induces Metabolic Remodeling of the Heart.

Androgen deprivation therapy of prostate cancer, which suppresses serum testosterone to castrate levels, is associated with increased risk of heart failure. Here we tested the hypothesis that castration alters cardiac energy substrate uptake, which is tightly coupled to the regulation of cardiac structure and function. Short-term (3-4 weeks) surgical castration of male mice reduced the relative heart weight. While castration did not affect cardiac function in unstressed conditions, we observed reductions in heart rate, stroke volume, cardiac output, and cardiac index during pharmacological stress with dobutamine in castrated vs sham-operated mice. Experiments using radiolabeled lipoproteins and glucose showed that castration shifted energy substrate uptake in the heart from lipids toward glucose, while testosterone replacement had the opposite effect. There was increased expression of fetal genes in the heart of castrated mice, including a strong increase in messenger RNA and protein levels of ß-myosin heavy chain (MHC), the fetal isoform of MHC. In conclusion, castration of male mice induces metabolic remodeling and expression of the fetal gene program in the heart, in association with a reduced cardiac performance during pharmacological stress. These findings may be relevant for the selection of treatment strategies for heart failure in the setting of testosterone deficiency.

Deficiency of mature B cells does not alter the atherogenic response to castration in male mice.

Testosterone deficiency in men is associated with increased atherosclerosis burden and increased cardiovascular risk. In male mice, testosterone deficiency induced by castration increases atherosclerosis as well as mature B cell numbers in spleen. As B cells are potentially pro-atherogenic, we hypothesized that there may be a link between these effects. To address whether mature B cell deficiency alter the atherogenic response to castration, we studied B cell-deficient µMT and genotype control male mice on an atherosclerosis-prone Apoe-/- background that were castrated or sham-operated pre-pubertally and fed a high-fat diet between 8 and 16 weeks of age to accelerate atherosclerosis development. Genotype did not affect the effects of castration on body weight or weights of fat depots and there were no differences in serum cholesterol levels across the four groups. Atherosclerosis assessed by quantification of lesion area in serial sections of the aortic root was significantly increased by castration and by the µMT mutation, with no significant interaction between genotype and surgery. In conclusion, castration evokes a similar atherogenic response in B cell-deficient µMT and control mice. These data suggest that atherogenesis following castration is unrelated to the effects of androgens on mature B cell numbers.

Low Progesterone and Low Estradiol Levels Associate With Abdominal Aortic Aneurysms in Men.

CONTEXT: Male sex is a major risk factor for abdominal aortic aneurysms (AAA) but few studies have addressed associations between sex hormone levels and AAA. OBJECTIVE: We aimed to describe the associations between serum sex steroids and early, screening-detected AAA in men. METHODS: We validated a high-sensitivity liquid chromatography-tandem mass spectrometry assay for comprehensive serum sex hormone profiling. This assay was then employed in a case-control study including 147 men with AAA (infrarenal aorta ≥ 30 mm) and 251 AAA-free controls recruited at the general population-based ultrasound screening for AAA in 65-year-old Swedish men. OUTCOMES INCLUDED: associations between dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, androstenedione, estrone, testosterone, dihydrotestosterone, and estradiol and AAA presence. RESULTS: Dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, testosterone, and estradiol, but not the other hormones, were lower in men with AAA. In models with adjustments for known AAA risk factors and comorbidity, only progesterone (odds ratio per SD decrease 1.62 [95% CI, 1.18-2.22]) and estradiol (1.40 [95% CI, 1.04-1.87]) remained inversely associated with the presence of AAA. Progesterone and estradiol contributed with independent additive information for prediction of AAA presence; compared with men with high (above median) levels, men with low (below median) levels of both hormones had a 4-fold increased odds ratio for AAA (4.06 [95% CI, 2.25-7.31]). CONCLUSION: Measured by a high-performance sex steroid assay, progesterone and estradiol are inversely associated with AAA in men, independent of known risk factors. Future studies should explore whether progesterone and estradiol, which are important reproductive hormones in women, are protective in human AAA.

Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway.

Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.

Androgen Receptors in Epithelial Cells Regulate Thymopoiesis and Recent Thymic Emigrants in Male Mice.

Androgens have profound effects on T cell homeostasis, including regulation of thymic T lymphopoiesis (thymopoiesis) and production of recent thymic emigrants (RTEs), i. e., immature T cells that derive from the thymus and continue their maturation to mature naïve T cells in secondary lymphoid organs. Here we investigated the androgen target cell for effects on thymopoiesis and RTEs in spleen and lymph nodes. Male mice with a general androgen receptor knockout (G-ARKO), T cell-specific (T-ARKO), or epithelial cell-specific (E-ARKO) knockout were examined. G-ARKO mice showed increased thymus weight and increased numbers of thymic T cell progenitors. These effects were not T cell-intrinsic, since T-ARKO mice displayed unaltered thymus weight and thymopoiesis. In line with a role for thymic epithelial cells (TECs), E-ARKO mice showed increased thymus weight and numbers of thymic T cell progenitors. Further, E-ARKO mice had more CD4+ and CD8+ T cells in spleen and an increased frequency of RTEs among T cells in spleen and lymph nodes. Depletion of the androgen receptor in epithelial cells was also associated with a small shift in the relative number of cortical (reduced) and medullary (increased) TECs and increased CCL25 staining in the thymic medulla, similar to previous observations in castrated mice. In conclusion, we demonstrate that the thymic epithelium is a target compartment for androgen-mediated regulation of thymopoiesis and consequently the generation of RTEs.

Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells-Brief Report.

OBJECTIVE: Androgen deprivation therapy has been associated with increased cardiovascular risk in men. Experimental studies support that testosterone protects against atherosclerosis, but the target cell remains unclear. T cells are important modulators of atherosclerosis, and deficiency of testosterone or its receptor, the AR (androgen receptor), induces a prominent increase in thymus size. Here, we tested the hypothesis that atherosclerosis induced by testosterone deficiency in male mice is T-cell dependent. Further, given the important role of the thymic epithelium for T-cell homeostasis and development, we hypothesized that depletion of the AR in thymic epithelial cells will result in increased atherosclerosis. APPROACH AND RESULTS: Prepubertal castration of male atherosclerosis-prone apoE-/- mice increased atherosclerotic lesion area. Depletion of T cells using an anti-CD3 antibody abolished castration-induced atherogenesis, demonstrating a role of T cells. Male mice with depletion of the AR specifically in epithelial cells (E-ARKO [epithelial cell-specific AR knockout] mice) showed increased thymus weight, comparable with that of castrated mice. E-ARKO mice on an apoE-/- background displayed significantly increased atherosclerosis and increased infiltration of T cells in the vascular adventitia, supporting a T-cell-driven mechanism. Consistent with a role of the thymus, E-ARKO apoE-/- males subjected to prepubertal thymectomy showed no atherosclerosis phenotype. CONCLUSIONS: We show that atherogenesis induced by testosterone/AR deficiency is thymus- and T-cell dependent in male mice and that the thymic epithelial cell is a likely target cell for the antiatherogenic actions of testosterone. These insights may pave the way for new therapeutic strategies for safer endocrine treatment of prostate cancer.

Prevalence and treatment of central hypogonadism and hypoandrogenism in women with hypopituitarism.

PURPOSE: Women with hypopituitarism have increased morbidity and mortality, and hypogonadism has been suggested to be a contributing mechanism. The purpose of this study was to investigate the prevalence of central hypogonadism and hypoandrogenism in women with hypopituitarism at a single Swedish center. METHODS: All consecutive women (n = 184) who commenced growth hormone (GH) replacement therapy at Sahlgrenska University Hospital in Gothenburg between 1995 and 2015 were included. In accordance with the Endocrine Society Clinical Practice Guidelines, strict criteria, based on menstrual history combined with laboratory measurements, were used to define central hypogonadism. Hypoandrogenism was defined as subnormal levels of dehydroepiandrosterone sulfate and/or androstenedione. RESULTS: Central hypogonadism was present in 78% of the women, in 75% of those ≤ 52 years and in 82% of those > 52 years of age. Hypoandrogenism was found in 61% of all the women and in 92% of those with adrenocorticotropic hormone (ACTH) deficiency. The estrogen substitution rate in hypogonadal women ≤ 52 years was lower than the hormonal substitution rate in the other pituitary hormone axes (74% versus 100%, P < 0.001). The use of estrogen substitution tended to decrease between 2000 and 2016. Few women received androgen treatment. CONCLUSIONS: In this first study of hypogonadism in women with hypopituitarism, using stringent diagnostic criteria for hypogonadism, the prevalence of central hypogonadism and low androgen levels was high and estrogen substitution was insufficient. Further studies are needed to elucidate the importance of hypogonadism and insufficient sex steroid replacement for the increased morbidity in hypopituitary women.

Testosterone is an endogenous regulator of BAFF and splenic B cell number.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

Dihydrotestosterone levels at birth associate positively with higher proportions of circulating immature/naïve CD5+ B cells in boys.

Boys present with higher proportions of immature/naïve CD5+ B cells than girls up to 3 years of age. Boys also have higher fractions of regulatory T cells (Tregs) in early infancy, but the mechanisms for these sex-related differences are unknown. In the prospective FARMFLORA follow-up study of 23 boys and 25 girls, we investigated if these immunological differences remained at 8 years of age. We also examined if testosterone or dihydrotestosterone (DHT) levels at birth and at 8 years of age were associated with immune maturation. Immunological variables and androgen levels were examined and measured in blood samples obtained at birth, 3-5 days and at 8 years of age. Boys had higher proportions of CD5+ and immature/transitional CD24hiCD38hi B cells, whereas girls had higher fractions of B cells with a memory phenotype at 8 years of age. School-aged boys also presented with higher frequencies of Tregs, and a greater capacity to produce T-cell-associated cytokines. Among boys, higher cord blood DHT levels were associated with higher proportions of CD5+ B cells in early infancy and at 8 years of life. These results suggest that DHT actions in utero might be involved in the mechanism for delayed peripheral B-cell maturation in boys.

Umbilical cord blood androgen levels in girls and boys assessed by gas chromatography-tandem mass spectrometry.

Androgen exposure of the fetus during gestation plays an important role in human physiology and pathophysiology, but assessment of androgens, in particular dihydrotestosterone (DHT), in human umbilical cord blood is technically challenging. The aim of this study was to assess umbilical cord androgen levels, including DHT, at birth by a highly sensitive assay, and study their association with sex of the infant, sex-hormone-binding globulin (SHBG) levels, and gestational age at delivery. Swedish infants (27 girls, 26 boys) were recruited at maternity care clinics in Southern Sweden. Umbilical cord blood levels of dehydroepiandrosterone (DHEA), androstenedione, testosterone and DHT at delivery were assessed by a gas chromatography-tandem mass spectrometry assay. Cord blood levels of DHT were 2.4-fold higher in boys (median 27.8pg/mL) than in girls (11.5pg/mL), while the sex difference was less pronounced for testosterone (1.3-fold higher in boys) and non-significant for DHEA and androstenedione. Gestational age at delivery associated inversely with DHT levels in boys and with DHEA levels in girls. There was a strong inverse correlation between SHBG and DHEA in both sexes, while there were no associations between SHBG and testosterone or DHT levels. In conclusion, using state of the art technology, we report that there is a pronounced sexual dimorphism in human umbilical cord blood DHT levels. The possibility to assess a complete androgen profile in human cord blood opens up for future increased understanding of the biological impact of the fetal androgen milieu.

The Bone Sparing Effects of 2-Methoxyestradiol Are Mediated via Estrogen Receptor-α in Male Mice.

2-Methoxyestradiol (2ME2), a metabolite of 17ß-estradiol (E2), exerts bone sparing effects in animal models. We hypothesized that the underlying mechanism is back conversion of 2ME2 to E2, which subsequently acts via estrogen receptor (ER)α. We measured serum E2 levels in orchidectomized wild-type (WT) mice treated with 2ME2 66.6 µg/d or placebo. In placebo-treated animals, E2 was below the detection limit. In 2ME2-treated mice, the serum E2 level was 4.97 ± 0.68 pg/mL. This corresponds to the level found in diesterus in cycling female mice. Next, we investigated bone parameters in orchidectomized WT and ERα knockout mice treated with 2ME2 or placebo for 35 days. 2ME2 (6.66 µg/d) preserved trabecular and cortical bone in WT mice. Trabecular volumetric-bone mineral density was 64 ± 20%, and trabecular bone volume/total volume was 60 ± 20% higher in the metaphyseal region of the femur in the 2ME2 group, compared with placebo (P < .01). Both trabecular number and trabecular thickness were increased (P < .01). Cortical bone mineral content in the diaphyseal region of the femur was 31 ± 3% higher in the 2ME2 group, compared with placebo (P < .001). This was due to larger cortical area (P < .001). Three-point bending showed an increased bone strength in WT 2ME2-treated animals compared with placebo (maximum load [Fmax] +19±5% in the 2ME2 group, P < .05). Importantly, no bone parameter was affected by 2ME2 treatment in ERα knockout mice. In conclusion, 2ME2 treatment of orchidectomized mice results in increased serum E2. ERα mediates the bone sparing effects of 2ME2. The likely mediator of this effect is E2 resulting from back conversion of 2ME2.

Enzalutamide Reduces the Bone Mass in the Axial But Not the Appendicular Skeleton in Male Mice.

Testosterone is a crucial regulator of the skeleton, but the role of the androgen receptor (AR) for the maintenance of the adult male skeleton is unclear. In the present study, the role of the AR for bone metabolism and skeletal growth after sexual maturation was evaluated by means of the drug enzalutamide, which is a new AR antagonist used in the treatment of prostate cancer patients. Nine-week-old male mice were treated with 10, 30, or 100 mg/kg·d of enzalutamide for 21 days or were surgically castrated and were compared with vehicle-treated gonadal intact mice. Although orchidectomy reduced the cortical bone thickness and trabecular bone volume fraction in the appendicular skeleton, these parameters were unaffected by enzalutamide. In contrast, both enzalutamide and orchidectomy reduced the bone mass in the axial skeleton as demonstrated by a reduced lumbar spine areal bone mineral density (P < .001) and trabecular bone volume fraction in L5 vertebrae (P < .001) compared with vehicle-treated gonadal intact mice. A compression test of the L5 vertebrae revealed that the mechanical strength in the axial skeleton was significantly reduced by enzalutamide (maximal load at failure -15.3% ± 3.5%; P < .01). The effects of enzalutamide in the axial skeleton were associated with a high bone turnover. In conclusion, enzalutamide reduces the bone mass in the axial but not the appendicular skeleton in male mice after sexual maturation. We propose that the effect of testosterone on the axial skeleton in male mice is mainly mediated via the AR.

Increased diet-induced fatty streak formation in female mice with deficiency of liver-derived insulin-like growth factor-I.

The role of endocrine IGF-I for atherosclerosis is unclear. We determined the importance of circulating, liver-derived IGF-I for fatty streak formation in mice. Mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice, serum IGF-I reduced by approximately 80 %) and control mice received an atherogenic (modified Paigen) diet between 6 and 12 months of age. At study end, Oil Red O staining of aortic root cryosections showed increased fatty streak area and lipid deposition in female but not in male LI-IGF-I(-/-) mice compared to controls. Mac-2 staining of aortic root and measurements of CD68 mRNA level in femoral artery revealed increased macrophage accumulation in proportion to the increased fatty streak area in female LI-IGF-I(-/-) mice. Moreover, female LI-IGF-I(-/-) mice displayed increased serum cholesterol and interleukin-6 as well as increased vascular cell-adhesion molecule 1 (VCAM1) mRNA levels in the femoral artery and elevated VCAM1 protein expression in the aortic root. Thus, increased diet-induced fatty streak formation in female LI-IGF-I(-/-) mice was associated with increased serum cholesterol and signs of systemic inflammation, endothelial activation, lipid deposition, and macrophage infiltration in the vascular wall.

Cardiovascular risk with androgen deprivation therapy for prostate cancer: potential mechanisms.

Androgen deprivation therapy (ADT) is frequently used for the treatment of advanced prostate cancer. ADT is associated with numerous side effects related to its mode of action, namely the suppression of testosterone to castrate levels. Recently, several large retrospective studies have also reported an increased risk of diabetes and cardiovascular disease in men receiving ADT, although these risks have not been confirmed by prospective randomized trials. We review the literature to consider the risk of cardiovascular disease with different forms of ADT and examine in detail potential mechanisms by which any such risk could be mediated. Mechanisms discussed include the metabolic syndrome resulting from low testosterone level and the potential roles of testosterone flare, gonadotropin-releasing hormone receptors outside the pituitary gland, and altered levels of follicle-stimulating hormone. Finally, the clinical implications for men prescribed ADT for the treatment of advanced prostate cancer are considered.

Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry.

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

Androgens regulate bone marrow B lymphopoiesis in male mice by targeting osteoblast-lineage cells.

Testosterone has profound immune-modulatory actions, which may be important for the sexual dimorphism in immune-related disorders, such as autoimmune diseases. A well-known effect of androgens is inhibition of bone marrow B lymphopoiesis; however, a plausible target cell for this effect has not yet been presented. The aim of this study was to determine the target cell for androgen-mediated regulation of bone marrow B lymphopoiesis in males. We confirm higher number of bone marrow B cells in male mice with global inactivation of the androgen receptor (AR) and these global AR knockout (G-ARKO) mice had increased number of B cell precursors from the pro-B stage. Because osteoblast-lineage cells are known to support B lymphopoiesis at the pro-B stage, we investigated the effect on B lymphopoiesis in osteoblast-lineage cell-specific ARKO (O-ARKO) mice; O-ARKO mice had increased number of B cells in the bone marrow, and the number of B cell precursors was increased from the pro-B stage, demonstrating that O-ARKO mimics the bone marrow B lymphopoiesis pattern of G-ARKO mice. By contrast, O-ARKO mice displayed only minor changes in B cell numbers in the splenic compartment compared with G-ARKO. Further, O-ARKO mice had moderately reduced number of bone trabeculae in the vertebrae, whereas cortical bone was unaffected. In conclusion, androgens exert inhibitory effects on bone marrow B lymphopoiesis in males by targeting the AR in osteoblast-lineage cells. The identification of the likely target cell for androgen-mediated regulation of bone marrow B lymphopoiesis will contribute to elucidation of the mechanisms by which androgens modulate immune-related disorders.